WO2012100598A1

WO2012100598A1 - Early-stage rheumatoid arthritis specific antigen peroxiredoxin iv

Info

Publication number: WO2012100598A1
Application number: PCT/CN2011/083629
Authority: WO
Inventors: 吴乔; 吴玉章
Original assignee: 中国人民解放军第三军医大学
Priority date: 2011-01-24
Filing date: 2011-12-07
Publication date: 2012-08-02
Also published as: CN102344916B; CN102344916A

Abstract

Disclosed in the present invention is an application of peroxiredoxin IV as a rheumatoid-arthritis specific antigen in the preparation of diagnostic reagents for rheumatoid arthritis, in particular the application of same in the preparation of diagnostic reagents for early-stage rheumatoid arthritis. Also disclosed is an application of peroxiredoxin IV in the preparation of a vaccine for rheumatoid arthritis.

Description

Rheumatoid arthritis early specific antigen peroxide reductase IV Technical field

The invention relates to the field of medicine, in particular to a rheumatoid arthritis-specific antigen.

Background technique

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by synovitis as a basic pathological change characterized by chronic destructive joint disease. Most of the conditions are progressive, eventually leading to joint fibrosis or bone. Sexually stiff and severely disabled, seriously affecting the quality of life of patients. Rheumatoid arthritis without proper treatment can be delayed and even cause joint deformities. Early detection and early treatment can effectively control the development of RA. Therefore, early diagnosis of RA is a problem of concern to scholars in various countries. Searching for serum markers with high specificity and sensitivity has attracted much attention.

In 2010, the American Association of Rheumatology (ACR) and the European Union Against Rheumatology (EULAR) proposed new RA classification criteria. It requires patients with a score to have at least one joint with clinically defined synovitis, and the cause of synovitis cannot be explained by other diseases. The criteria are based on the integration of the affected joint, serum antibody test results, acute phase reactants, duration of symptoms, and other items. A total score of 10 points and 6 points will confirm the diagnosis of RA; less than 6 points can not confirm the diagnosis of RA, but patients may meet the diagnostic criteria in the future.

In terms of experimental diagnosis, in addition to the past rheumatoid factor (RF), there are antibodies against citrullinated peptide (CCP), anti-RA33 antibody, anti-keratin antibody, anti-peripherin antibody. The positive rate of RF in RA can be as high as 75%-80%, but its specificity is poor. RF can also be detected in other diseases, such as systemic lupus erythematosus, malaria, tuberculosis, etc.; even in normal human serum. Detected (positive population positive rate 3%-5%, elderly 10%-30%); RF positive results may also occur after some vaccinations. The target antigen against the RA-33 antibody is a 33 kD nucleic acid protein, which is a specific antibody produced against the nuclear protein of Hela cells and cross-reacts with hnRNP. Anti-RA-33 can occur in early RA, which is beneficial for the early diagnosis of RA. The antibody is highly specific, up to 99%, but with a sensitivity of only 35.8%. Usually RA33 always appears simultaneously with RA36, which is more specific. The sensitivity of RA patients in China is 28.9%-37.7%, and its specificity is 89.8%-95.5%. Because anti-RA33 antibodies are also detected in the serum of approximately one-third of patients with systemic lupus erythematosus and mixed connective tissue disease, the specificity of anti-RA33 antibodies for RA diagnosis is questioned. The sensitivity and specificity of anti-CCP antibodies were 71.4% and 92.5%, respectively. However, among patients who meet the diagnostic criteria for RA classification, there are still a significant number of patients with negative anti-CCP antibodies. Correlation studies between peroxide reductase IV and rheumatoid arthritis have not been reported.

Summary of the invention

In view of the above, one of the objects of the present invention is to provide a novel application of a peroxide reductase IV by screening RA candidate antigens in RA synovial tissue and screening out the candidate antigens. Oxide reductase IV and The antibodies and immune complexes were tested for specificity and sensitivity by ELISA in the serum of patients with early and late RA. Peroxidase IV was mainly expressed in the cytoplasm of synovial cells at the beginning of RA lesions, peroxide reductase IV. In vitro specific stimulation of peripheral blood mononuclear cells (PBMC) in RA patients proliferate and secrete RA-associated cytokines IL-17A, TNF-α and IFN-γ, so peroxide reductase IV is a new RA disease-associated antigen. The new application of antigen-antibody and its immune complex detection has high specificity and sensitivity in the serum of early RA patients, which provides a new idea for the early diagnosis and treatment of rheumatoid arthritis.

To achieve the above object, the technical solution of the present invention is:

The use of peroxide reductase IV in the preparation of rheumatoid arthritis specific antigens.

Further, the use of the peroxide reductase IV antigen, the epitope peptide, the immune complex and the antibody thereof for preparing a diagnostic reagent for rheumatoid arthritis;

Further, the use of the peroxide reductase IV antigen, the epitope peptide, the immune complex and the antibody thereof for preparing an early diagnostic reagent for rheumatoid arthritis;

Further, the use of peroxide reductase IV in the preparation of a rheumatoid arthritis vaccine.

The technical scheme uses the immunoproteomics method to screen the RA candidate antigen and antibody in the RA synovium, and further identifies and verifies the fifth of the candidate antigen by TOF-TOF mass spectrometry and Western Blotting. The protein spot was peroxide reductase IV; the expression of peroxide reductase IV in the synovial tissue of RA and osteoarthritis (OA) was detected by Western-blot; the peroxide reductase IV was confirmed by immunohistochemical staining. It is mainly expressed in the cytoplasm of synovial cells in the initial lesion of RA; the specificity and sensitivity of peroxiredoxin IV antibody in RA serum were determined by indirect ELISA; peroxide reductase IV was determined by double antibody sandwich ELISA The specificity and sensitivity of its immune complexes in RA serum; it was found that peroxide reductase IV antibody and immune complexes were significantly higher in patients with advanced RA (more than 2 months), and peroxide reduction Enzyme IV was significantly higher in patients with early RA (less than 2 months) and was higher than patients with advanced RA who were treated with immunosuppression; It was confirmed by cell proliferation experiments that peroxidase reductase IV specifically stimulates proliferation of peripheral blood mononuclear cells of RA, and secretes IL-17, TNF-α and IFN-γ and other cytokines associated with RA. Based on the above results, the present invention concludes that the peroxide reductase IV is an RA-specific disease-associated antigen. The preparation of vaccines based on the above studies on the modification of the peroxide reductase IV antigen may provide a new means for specific immunotherapy of RA.

The beneficial effects of the invention are as follows: The use of peroxide reductase IV in the preparation of rheumatoid arthritis-specific antigens has high specificity and sensitivity, and is especially suitable for the application of early diagnostic reagents, and the peroxide reductase IV is also New ideas for the preparation of rheumatoid arthritis vaccines.

DRAWINGS In order to make the objects, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings, in which:

Figure 1 shows the two-dimensional gel electrophoresis pattern of Coomassie blue staining after 2-D separation of RA synovial tissue protein spots.

Figure 2 is a two-dimensional gel electrophoresis pattern of the hybridization of serum from RA patients to the protein spots of Figure 1.

Figure 3 is a two-dimensional gel electrophoresis pattern of OA patient sera hybridized with Figure 1 protein spots.

Figure 4 is a two-dimensional gel electrophoresis pattern of normal human serum hybridized with the protein spots of Figure 1.

Figure 5 is a two-dimensional gel electrophoresis map of the serum of systemic lupus erythematosus (SLE) patients hybridized with the protein of Figure 1.

Figure 6 is a two-dimensional gel electrophoresis map of the specificity of RA synovial tissue in response to serum from RA patients, containing 12 protein spots.

Figure 7 is a map of the No. 5 protein spot of TOF-TOF protein tandem mass spectrometry.

Figure 8 is a Western blotting protein qualitative detection electrophoresis spectrum, where A is a one-way gel electrophoresis map (1, 2 is different RA synovial tissue, 3, 4 is Hela cell positive control), B is RA synovial tissue bidirectional condensation Gel electrophoresis map.

Figure 9 shows the differential expression of peroxisome IV in the synovial tissue of RA patients and patients with OA by immunoblotting (1, 2 for RA and OA synovial tissue, respectively).

Figure 10-A and Figure 10-B show pathological photographs of RA synovial tissue stained with hematoxylin-red (HE), and Figure 10-C and Figure 10-D for tissue synthase reductase IV immunohistochemistry of RA synovial tissue A pathological photo of the stain.

Figure 11 is a graph showing the stimulation index (SI) of the PBMC proliferation experiment.

Figure 12 shows the proliferation of PBMC by CFSE staining flow cytometry.

Figure 13 is a graph showing the analysis of the amount of IFN- y, IL-17 and TNF-α secreted by PBMC by ELISA.

detailed description

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Experimental Materials

1.1 reagent

Acrylamide, methylene acrylamide, TEMED, ammonium persulfate, sodium dodecyl sulfate, Tris, glycine, bromophenol blue, dithiothreitol (DTT), CHAPS, low melting agarose, low molecular weight standard protein , IPG buffer, IPG strip (pH 3-ll L), IPG overlay oil, urea and PhastGel Blue R were purchased from Amersham Pharmacia Biotech; iodoacetamide was purchased from Sigma; sequencing grade trypsin was purchased from Promega; protease inhibitor and ECL Western blot detection kit was purchased from Roche; methanol, glacial acetic acid and absolute ethanol were purchased from Chongqing Chuandong Chemical (Group) Co., Ltd. Chemical Reagent Factory; horseradish-labeled goat anti-human IgG (H+L), horseradish enzyme label Goat anti-mouse IgG ( H+L ), Power VisionTM (IgG antibody-HRP multimer) complex and DAB chromogenic kit were purchased from Beijing Zhongshan Biotechnology Co., Ltd.; mouse anti-human peroxide reductase IV monoclonal antibody was purchased from Abeam (potency 1:12800); Recombinant human peroxide reductase IV was purchased from Lifescience; paraformaldehyde was purchased from Beijing Chemical Reagent Company.

1.2 Instrument

IPGphor isoelectric focusing instrument, SE600 vertical electrophoresis instrument, MultiTempIII circulating water bath and TE50X electrorotator were purchased from Amersham Pharmacia Biotech; 550 enzyme enzyme analyzer and GS-800 image scanning system Bio-Rad; MALDI-TOF mass spectrometer was purchased from Bruker The company; the cryogenic high-speed centrifuge was purchased from Wuhan Scientific Instrument Factory of Chinese Academy of Sciences; the bleaching shaker was purchased from Jiangsu Haimen Qilin Bell Instrument Manufacturing Co., Ltd.; the LEICA AM 2135 slicer was purchased from Leica; BX-50 Olympus microscope and PM- Type 20 Olympus microscopic camera system Olympus; UV spectrophotometer Backmen.

1.3 main reagent preparation

1.3.1 Sample lysate stock solution: 40 mmol/L Tris, 7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 1 ml/tube, and frozen at -70 °C.

1.3.2 lOmg/ml RNase A, -20 °C frozen for use.

1.3.3 mg/ml DNase I, stored at -20 °C for later use.

1.3.4 20 X protease inhibitor: One protease inhibitor was dissolved in 350 μL of pure water.

1.3.5 1 mol/L DTT, stored at -20 °C for later use.

1.3.6 IPG buffer, 4 °C save standby.

1.3.7 Sample lysate working solution: 40mmol/L Tris, 7 mol/L urea, 2mol/L thiourea, 4% CHAPS, 100 μg/ml DNase I, 25 μg/ml RNase A, IX protease inhibitor, 0.8% IPG buffer, 60 mmol/L DTT.

1.3.8 Solution liquid: 7 mol/L urea, 2 mol/L thiourea, 2% CHAPS, trace bromophenol blue, frozen at -70 °C.

1.3.9 Soluble liquid working solution: Add 1% IPG buffer, 60 mmol/L DTT (pre-use plus) to the stock solution.

1.3.10 Equilibration buffer: 50 mmol/L Tris-Cl H 8.8, 6 mol/L urea, 30% glycerol, 2% SDS, bromophenol blue trace.

1.3.11 The mass fraction is 0.5% low melting point agar: The low melting point agar with a mass fraction of 0.5% is dissolved in SDS-PAGE running buffer.

1.3.12 30% acrylamide stock solution: 30g acrylamide, 0.8g of fork acrylamide dissolved in 100ml of pure water, Filter, store at 4 ° C in a brown bottle.

1.3.13 4 X Separation Gum Buffer: 1.5 mol/L Tris-Cl (pH 8.8)

1.3.14 10 SDS: lOg SDS was dissolved in 50 ml H ₂ O to a volume of 100 ml.

1.3.15 10% ammonium persulfate: O.lg ammonium persulfate is dissolved in 1 ml of pure water.

1.3.16 SDS-PAGE running buffer: 94 g glycine, 12.1 g Tris, 50 ml 10 % SDS, dissolved in water and made up to 1000 ml.

1.3.17 Coomassie Brilliant Blue Stain Solution: Dissolve 1 piece of PhastGel Blue R in 80ml of water, stir 5- lOmin, add 120mL of methanol, stir until the dye is completely dissolved, and filter the solution for later use.

1.3.18 Coomassie Brilliant Blue Staining Solution (0.1%): The solution of the staining solution is mixed with a volume fraction of 20% acetic acid in a ratio of 1:1 by volume.

1.3.19 Decolorizing solution: The volume fraction is 30% ethanol, and the volume fraction is 10% acetic acid dissolved in water.

1.3.20 Transfer buffer (39mm glycine, 48mm Tris base, mass fraction of 0.037% SDS, volume fraction of 20% methanol): Weigh 2.9g of glycine, 5.8g of Tris base, 0.037g of SDS, and add 200ml of methanol, finally Add water to the total volume of 1000ml.

1.3.21 TBS (50 mM Tris base, 150 mM sodium chloride, ρΗ7·5): Weigh 6.05 g of Tris base, 8.76 g of sodium chloride, adjust the pH to 7.5 with HC1, add water to a total volume of 1 L, 2-8 Store at °C for 3 months.

1.3.22 TBST: Add lmLTween 20 to 1L TBS and store at 2-8 °C for 3 months.

1.3.23 1% blocking solution: Add 10mL blocking solution to 90ml TBS and store at -15~-25 °C.

1.3.24 0.5% blocking solution: Add 5mL blocking solution to 95mL TBS and store at -15~-25 °C.

1.3.25 Primary antibody (serum): Dilute primary antibody with 0.5% blocking solution.

1.3.26 POD-labeled anti-mouse or rabbit IgG: The lyophilized powder was dissolved in 100 μL of pure water to obtain a secondary anti-liquid. Dilute with 0.5% blocking solution to obtain a working solution. A 40 mU/mL secondary antibody is usually sufficient for detection. The stock solution can be stored for 12 months at 2-8 °C, but the working fluid needs to be freshly configured.

1.3.27 ECL test solution: Mix solution A and solution B in a ratio of 1:1 by volume. The solution is ready for use.

1.3.28 0.01 M PBS: 50 mL of 0.2 mol/L PB plus 8.8 g of NaCl, and make up to 1000 mL with double distilled water.

1.3.29 RIPA lysate: 1.5M Nacl lmL, lOOmM Tris-HCL (pH 7.4) 5mL, add mass fraction of 10 SDS lOOuL, 500mM DTT lOOuL, NP-40 lOOuL, Complete Mini (protease inhibitor) 1 tablet, Three distilled water to a volume of 10 mL.

1.3.30 coating solution: 0.85M (pH 9.6) carbonate buffer: 1.59g Na ₂ C0 ₃ force B 2.93g NaHC0 ₃ , three steam The volume of water is 1000 mL.

1.3.31 Washing solution: 0.01M pH 7.4 PBS Ten Tween -20 (T) The final mass fraction is 0.05%.

1.3.32 substrate solution: phosphate-citric acid buffer (pH 5.0~5.5): 0.1M citric acid 24.3mL added 25.7ml

0.2M Na ₂ HP0 ₄ · 12H ₂ 0 Add 40.0 mg of o-phenylenediamine, and make up to 50 mL. Add 0.15 mL volume fraction before use.

30% of 3⁄40 ₂ .

1.3.33 Terminator: 22.2 mL of sulfuric acid was added to distilled water 177.8 mL.

1.4 Samples: 5 cases of RA synovial tissue were provided by Professor Li Zhanguo from the Department of Rheumatology and Immunology, Peking University Affiliated People's Hospital; 534 cases of RA (67 cases in early stage, 467 cases in advanced stage), 65 cases of OA, 120 cases of SLE, 10 cases of scleroderma ( Scleroderma ), 15 cases of dermatomyositis and 120 normal controls (NC) Blood samples were obtained from Southwest Hospital.

2. Two-dimensional gel electrophoresis for screening candidate antigens in RA synovial tissue

2.1 Sample preparation

2.1.1 Remove the RA synovial tissue sample frozen at -70 °C, equilibrate at room temperature for about 10 min, add the sample lysate working solution to grind well, shake on the vortex for about 20-30 min, and let stand at room temperature for l-2 h to make the cells The protein was fully dissolved and subjected to sonication at 12000 rpm for 1 hour at 4 °C.

2.1.2 Protein quantification of the sample solution (DC Protein Assay, BIO-RAD COMPANY).

2.1.2.1 Prepare protein standard solution (0.25mg/ml~1.5mg/ml protein): Dissolve BSA in water to prepare 10mg/mL BSA mother liquor, then dilute it to 0, 0.25, 0.5, 1.0, 1.5 with sample lysate. Mg/ml five different concentrations of BSA solution.

2.1.2.2 Dilution of sample solution: Dilute the sample solution with sample lysate 10, 20 times for use.

2.1.2.3 Prepare the working fluid reagent Α' : Add 20 μL of reagent S per 1 ml of reagent A (The working solution reagent A' is stable for 1 week, even after 1 day, if a precipitate forms, heat the solution and oscillate. Do not blow with a pipette tip, as it is easy to block the tip and change the volume of reagent added to the sample).

2.1.2.4 Samples and Standards Α 655 value determination: Pipette 5 μL of standard solution and sample into a clean, dry microplate. Add 25 L of reagent A' or reagent 每 to each well. Add 200 L of reagent B to each well. After 15 min, the absorbance was measured at 655 nm. The absorbance value in lh is stable.

2.1.2.5 Establish a linear equation and calculate the sample protein concentration.

2.2 Experimental methods

2.2.1 2D-PAGE first direction - isoelectric focusing.

2.2.1.1 Remove the IPG strip frozen at -20 °C and equilibrate for several minutes at room temperature. 2.2.1.2 Mix the sample and the swelling solution in the IPG holder to achieve the proper loading. Remove the protective film of the IPG strip with the adhesive side facing down to avoid air bubbles and put it into the swelling solution. The IPG strip is covered with a layer of mineral oil, capped and allowed to swell overnight.

2.2.1.3 The operating parameters of the instrument are as follows:

Isoelectric focusing operating parameters: temperature, 20 °C; maximum current, 50 mA per IPG strip; sample volume, 350 μ 1 (180 mm IPG strip) or 250 l (130 mm IPG strip); 30 V, 10-12 hours (melting) 200V, 1 hour; 500V, 1 hour; 500-8000V, 30min; 8000V, 5-6 hours (IPG 3-10L), 6-7 hours (IPG 4-7).

2.2.1.4 After reaching the appropriate total voltage-time product (Total Volt-hours, Vh), remove the strip and equilibrate in the equilibration solution.

2.2.2 Balance

2.2.2.1 lOOmg DTT was dissolved in 10 mL of equilibration buffer (Equilibration Buffer I). The IPG strips were taken out and placed in a glass tube and shaken on the shaker for 15 min.

2.2.2.2 Dissolve 400 mg of iodoacetamide in 10 mL of equilibration buffer (Equilibration Buffer II), transfer the strip to Equilibrium Buffer II, cap and shake for 15 min on the shaker.

2.2.3 2D-PAGE second direction - SDS-PAGE

2.2.3.1 Glue filling

Gum concentration 10 % 12.5 %

30% acrylamide stock solution 33.3ml 41.7 ml

1.5M Tris-HCl 25 ml 25 ml

10 % SDS 1 ml 1 ml

Pure water 40.2 ml 31.8 ml

10% ammonium persulfate 500μ1 500μ1

TEMED 33μ1 33 μΐ

Total volume 100 ml 100 ml

2.2.3.2 Install the glue film according to the instrument manual, pour the gel solution, add pure water to each piece of glue to obtain a flat gel loading plane. Polymerize for at least 2 h at room temperature.

2.2.3.3 The electrophoresis tank is filled with electrophoresis buffer, and the thermostatic water bath system is turned on, and the temperature is adjusted to 15 °C.

2.2.3.4 Immerse the well-balanced IPG strip in pure water for 1 second to remove excess equilibration buffer and place the strip on the filter paper to remove excess water.

2.2.3.5 Carefully place the IPG strip on the SDS surface and gently press the IPG strip to the SDS surface to cover the 0.5% low melting point agarose solution to allow the agarose to solidify within 5 minutes.

2.2.3.6 Insert the plastic box into the electrophoresis tank and start electrophoresis.

2.2.3.7 SDS electrophoresis operating conditions: Use SE600 electrophoresis instrument, the specific method is as follows: temperature, 15 ° C; current time, 10 mA / glue, 15 min; 25 mA / glue, 3-4 hours. 2.2.3.8 Electrophoresis is terminated when bromophenol blue migrates to the bottom edge of the gel.

2.3 staining and image analysis

2.3.1 Dyeing

After electrophoresis, freshly prepared Coomassie Brilliant Blue staining solution (0.1%) was used, stained for 4 h or overnight, and destained with decolorizing solution until the background was clean.

2.3.2. Gel image acquisition and image analysis

Two-dimensional electrophoresis gel scanning was performed using a GS-800 image analysis system, and gel image analysis was performed using PDQuest 7.0 software (Bio-Rad).

2.4Western blot

2.4.1 Transfer process:

2.4.1.1 After the SDS electrophoresis is completed, mark the chamfered corner and equilibrate the gel at room temperature for 15 min with an appropriate amount of transfer buffer.

2.4.1.2 Put on gloves, cut 6 sheets of filter paper and 1 PVDF membrane, the same size as the glue, and soak the filter paper in the transfer buffer.

2.4.1.3 Wet the PVDF membrane with 100% methanol for 15 seconds, then dip into pure water for 3 min, and finally equilibrate for 15 min in transfer buffer.

2.4.1.4 Assemble the transfer sandwich, from the negative electrode to the positive electrode: Filter paper - gel - PVDF film - filter paper.

2.4.1.5 Place the sandwich in the transporter and turn on the power according to the gel area of 0.65 mA/cm ² . The electrical transfer time is determined according to the specific conditions.

2.4.1.6 After transshipment, stain with Ponceau S.

2.4.1.7 After the PVDF membrane is fully rinsed, specific antibody hybridization is carried out to develop color.

2.4.1.8 Sealing of the membrane: Place the membrane in a plastic bag, add 1% blocking solution according to the membrane area, and block at 4 °C overnight. 2.4.1.9 Incubation of Primary Antibody with PVDF Membrane: Incubate serum or normal human serum of colorectal cancer with PVDF membrane at a concentration of 1:200.

2.4.1.10 Rinsing: TBST was washed twice, each time lOmin; 0.5% blocking solution was washed twice, each for 10 min. 2.4.1.11 Incubation of POD-labeled secondary antibody with PVDF membrane: Add the secondary antibody dilution solution according to the membrane area, and incubated on a gently shaking platform for 30 min at room temperature.

2.4.1.12 Rinse: Wash 4 times with a large amount of TBST for 15 min each time.

2.4.2 Color development process: After exposing the film to the detection reagent, the color reaction must be carried out quickly to avoid the luminescence reaction subsiding. All processes must be carried out in a dark room. 2.4.2.1 Fill a clear, transparent plastic bag of the same size as the film, then place the film in the bag and gently roll it with a 10 ml glass pipette to drive out excess liquid.

2.4.2.2 Add pre-mixed test solution.

2.4.2.3 Insert the membrane into the colorimetric box with the protein side facing up.

2.4.2.4 Turn off the light, place a film on the film, and close the color box. Exposure 10-60s.

2.4.2.5 Immediately change a new film on the film, close the color box, and color the film just exposed. During the color development process, red light monitoring can be turned on.

2.4.2.6 Predict the appropriate exposure time of the second film from the signal intensity of the first film. The luminescence reaction peaked after 1 to 2 minutes and lasted for 20-30 minutes. After 1 hour, the signal intensity decreased to 60-70% of the peak. If the signal is too strong, you can wait for lOmin for a second exposure.

Conclusion: Immunohistochemical method was used to screen 12 synovial tissue protein spots that specifically react with RA serum by setting OA, normal human and SLE serum control as RA candidate antigens. See Figure 1 6.

3. Identification of peroxide reductase IV and its differential expression and pathological tissue distribution from RA candidate antigens

3.1 MALDI-TOF/TOF mass spectrometry identification

3.1.1 Enzymatic digestion

3.1.1.1 Decolorization of Coomassie Brilliant Blue Stained Gel: Cut the protein spots in the gel after electrophoresis staining, cut the gel into l-2mm ² size with a clean scalpel, and wash it with ultrapure water several times. Soak the glue point with 50% acetonitrile, 25 mmol/L NH ₄ HC0 ₃ solution, shake for 20 min, discard the solution, repeat 1~2 times until the blue point in the glue point is exhausted.

3.1.1.2 Digestion: Vacuum drying by vacuum for about 20 minutes, so that the film is completely dehydrated and reduced in volume. Add 5~10 μ1 trypsin solution (0.01 g/l, containing 25 mmol/L NH ₄ HC0 ₃ ), and place at 20 °C at 20 °C. After 30 min, the enzyme solution was completely absorbed, supplemented with 5-10 μ 1 25 mmol/L NH ₄ HC0 ₃ and incubated at 37 ° C for 15 h.

3.1.1.3 peptide extraction: add 5% TFA 50~100 μ 1 at 40 ° C for 1 h, aspirate the supernatant, add 2.5% TFA, 50% acetonitrile 50-100 μ 1 at 30 ° C for 1 h, aspirate the supernatant, The supernatants were combined and lyophilized. After drying, the sample was dissolved in 3 to 5 μl of a 0.5% TFA solution. The sample was desalted using a C18 Ziptip (Millipore, USA) and the desalting procedure was followed according to the instructions.

3.1.2 MALDI-TOF/TOF analysis: The enzyme was cut into samples and sent to Beijing Huada Genomics Co., Ltd. for analysis by MALDI-TOF/TOF mass spectrometry.

3.1.3 Database query and parameter setting:

As shown in Figure 7, the maximum allowable peptide mass error is: 400 ppm; one incomplete cut is considered for each peptide; the isoelectric point range is: 3-10; sequence homology analysis was performed using Mascot and NCBI BLAST. 3.2 Determination of mass spectrometry by peroxide reductase IV monoclonal antibody gel electrophoresis

One-dimensional verification: The frozen tissue was taken out from liquid nitrogen, and accurately weighed and added to the RIPA lysate at l:10 (w/v). The homogenate was thoroughly ground in an ice bath, and the homogenate was sucked into a 1.5 mL centrifuge tube and lysed on ice for 60 min. Centrifuge at 4 ° C, 12000 g X 60 min. The supernatant was taken and dispensed, and stored at -70 °C for later use. Protein quantification of the sample solution (same method as described above). Prepare 10% separation gel and 5 % laminating gel; Sample preparation before loading: Adjust the protein concentration according to protein quantification, the protein sample loading is 50mg (total volume 20mL). Electrophoresis: The laminated glue voltage is 80V, and the separation glue voltage is changed to 120V. Abeam mouse anti-human peroxide reductase IV monoclonal antibody was used as the primary antibody, and Beijing Zhongshan Biotechnology Co., Ltd. horseradish-labeled goat anti-human IgG (H+L) was used as secondary antibody, electroporation, hybridization, and development. The aforementioned method. The results are shown in Figure 8.

Results: Figure 8-A shows the results of one-dimensional verification mass spectrometry identification of peroxide reductase IV monoclonal antibody. 1, 2 are synovial tissue of two different RA patients respectively; 3, 4 are positive control of Hela cells; thus confirming mass spectrometry identification The No. 5 protein spot was expressed in different RA synovial tissues.

Two-dimensional verification: RA tissue protein spots were isolated by the same method as the above two-dimensional gel electrophoresis, and ABL mouse anti-human peroxide reductase IV monoclonal antibody was incubated after transfection, and the protein spots appeared after hybridization with RA serum after development. Position comparison.

Results: Figure 8-B shows the results of two-dimensional verification mass spectrometry of peroxide reductase IV monoclonal antibody. The reaction site of peroxide reductase IV monoclonal antibody and RA synovial tissue is the same as the position of point 5 of RA serum reaction in Figure 2; It is indicated that a peroxide reductase IV antibody that reacts with synovial tissue is indeed present in the RA serum.

3.3 Differential expression of peroxide reductase IV in RA and OA synovial tissue

SDS-PAGE electrophoresis: RA and OA protein samples were prepared in the same manner as in 3.2. Electrophoresis: The laminated glue voltage is 80V, and the separation glue voltage is changed to 120V. The steps of electroporation, hybridization, and development are the same as those described above. The results are shown in Figure 9.

Results: Figure 9 shows that the expression of peroxide reductase IV in RA synovial tissue is significantly higher than that of ΟΑ, 1, 2 and RA and OA synovial tissues, respectively. The upper band is β-actin internal reference (42Kd), and the lower part is for the purpose. Strip (31Kd);

3.4 Distribution of peroxide reductase IV in diseased tissues

Immunohistochemical staining was performed using the Power Vision TM two-step method, which was operated according to the instructions. The specific methods are as follows:

3.4.1 Paraffin-embedded tissue sections (4 μ ηι~5 μ ηι), heated in a 60 °C oven for 24 h.

3.4.2 Dewaxing of xylene, gradient hydration of ethanol. The pure xylene was dewaxed twice (5 min, 7 min, respectively), and 100%, 100%, 95%, 80%, 70% gradient alcohol was hydrated to water.

3.4.3 Soak in PBS for 5 min X 2, add 3 % methanol-H ₂ 0 _{2 and} block at room temperature for 10 min to eliminate endogenous peroxidase. 3.4.4 Soak 5 min X 3 in PBS, microwave antigen retrieval, soak in 0.01 M citrate buffer (pH=6), heat in microwave oven, mid-range 6 min, intermittent 2 min, low grade 5 min, cool at room temperature for 20 min.

3.4.5 5 % normal goat serum was blocked at room temperature for 10 min.

3.4.6 Pour off the serum, add 1:50 diluted peroxide reductase IV monoclonal antibody, and store in a humid box at 4 ° C overnight. The primary antibody was replaced with PBS as a negative control.

3.4.7 Soak in PBS, 5 min X 3, add Power VisionTM Complex 50 μL, incubate at 37 °C, 30 min.

3.4.8 Soak in PBS, 5 min X 3, DAB color development, use DAB color development kit, add one drop of A, B, C solution to 0.85ml distilled water, mix well, add to slide, and develop color for 10min at room temperature.

3.4.9 Washing with tap water, hematoxylin counterstaining 15s~30s, 1% hydrochloric acid alcohol differentiation, saturated lithium carbonate blue.

3.4.10 Conventional gradient alcohol dehydration, xylene transparent, DPX seal storage.

3.4.11 Observing under the microscope, taking pictures. The results are shown in Figure 10.

Figure 10-A, B (100 X and 200 X, respectively) showed HE staining of RA synovial tissue, which showed synovial cell hyperplasia, a large number of inflammatory cells infiltrated in synovial tissue, mainly lymphocytes; Figure 10-C, D (100 X and 200 X, respectively) showed the distribution of peroxide reductase IV in synovial tissue. Immunohistochemical staining confirmed that peroxide reductase IV was mainly expressed in synovial cell cytoplasm;

3.5 Peroxidase reductase IV antigen specific stimulation in vitro RA peripheral blood mononuclear cell proliferation

3.5.1 ³ H-TdR incorporation method

Lymphocytes were isolated from RA, SLE, dermatomyositis, scleroderma, OA, and normal human peripheral blood mononuclear cells, and cultured in 96-well conical plates at 2 X 10 ⁵ /well. Unstimulated, peroxide reductase IV antigen stimulation (50 ug/mL) and PHA-stimulated positive control group were separately set and cultured for 3 days. Each group of cells was spiked with ³ H-TdR 3.7 X 10 ⁶ Bq/well ^{18 to 24} h before termination of culture, and the culture was terminated after reaching the corresponding observation time. The supernatant was discarded, and the sample was collected on a glass fiber filter paper using a multi-head cell harvester. , drying. The filter papers collected with different samples were respectively clamped into the liquid flash bottle, and the scintillation liquid was added, and the pulse count value per minute (cpm) was measured in the β liquid scintillation meter. The PBMC proliferation stimulation index (SI = test sample cpm / negative control cpm) (see Figure 11) is indicated.

3.5.2 CFSE labeling flow cytometry detection PBMC proliferation

The number of peripheral blood mononuclear cells in freshly isolated RA patients and each control group was adjusted to 8×10 ⁶ /L, and after centrifugation at 700 g X for 5 minutes, it was resuspended in 1 ml of 0.01 M PBS containing 5% FBS. CFSE (storage concentration 10 mmol/L) was diluted to 5 mmol/L with DMSO, and llul CFSE working solution was added to llOul 0.01 M PBS, added dropwise to the cells, mixed, and incubated at 37 ° C for 10 min. Thereafter, the reaction was stopped by adding 10 ml of 0.01 M PBS containing 5% FBS. The supernatant was centrifuged at 700 g for 5 minutes, and the above operation was carried out three times. The cells were suspended in RPMI1640 complete medium, and the number of cells was adjusted to 2×10 ⁵ /L. Connect 96-well plates, 200 Lo per well, each with 3 peroxide reductase IV stimulating wells (50 ug/well), 3 negative control wells (50 ul 0.01 M PBS) and 3 PHA stimulating wells (25 mg) /L), continue to culture for 3 days at 37 ° C, 5% CO ₂ . Each sample was analyzed for 10,000 cells using a flow cytometer (Beckman FC500). The acquired data was analyzed by Cell Quest software. The lymphocyte region is first drawn by a two-dimensional scatter plot of front scatter (FS ) and side scatter (SS ). The results obtained are shown in a histogram, as shown in Figure 12.

Conclusion: ³ H-TdR incorporation and CFSE labeling flow cytometry have simultaneously confirmed that peroxiredoxin IV antigen specifically stimulates peripheral blood mononuclear cell proliferation in vitro, indicating that peroxide reductase IV is specific for RA. Sex antigen. 3.6 Peroxired reductase IV antigen specifically stimulates peripheral blood mononuclear cells from IL to secrete IL-17A, TNF-α and INF-γ

Freshly isolated RA patients and peripheral blood mononuclear cells of each control group were cultured in 48-well plates at 1×10 ⁶ /L per well. Each sample was provided with 3 peroxide reductase IV stimulation wells (50 ug/well). Three negative control wells (50ul 0.01M PBS) and three PHA stimulation wells (25 mg/L) were cultured for 3 days at 37 °C and 5% C0 ₂ . Centrifuge at 12000 rpmX for 5 minutes at 4 ° C, and collect the supernatant. The pre-packaged human IL-17A, TNF-α and INF-Y ELSIA kit instructions were used as follows: 50 ul of the diluted standard was added to the reaction well, and 50 ul of the sample to be tested was added to the reaction well. Immediately add 50 ul of biotinylated antibody. Cover the membrane, mix gently by shaking, and incubate at 37 °C for 1 hour. Remove the liquid from the well, fill each well with the washing solution, shake for 30 seconds, remove the washing solution, and pat dry with absorbent paper. Repeat this operation 3 times. If washing with a washer, the number of washings is increased once. 80 ul of affinity streptavidin-HRP was added to each well, and the mixture was gently shaken and incubated at 37 ° C for 30 minutes. Remove the liquid from the well, fill each well with the washing solution, shake for 30 seconds, remove the washing solution, and pat dry with absorbent paper. Repeat this operation 3 times. If washing with a washer, the number of washings is increased once. Add 50 L of each of the substrates A and B to each well, mix gently by shaking, and incubate at 37 V for 10 minutes. Avoid lighting. Remove the plate and quickly add 50 L of stop solution. Immediately after adding the stop solution, the results should be determined. The OD value of each well was measured at a wavelength of 450 nm. According to the standard curve, the linear regression equation is calculated, and finally the concentration of each cytokine in the test sample is obtained. As shown in Figure 14, A, B, and C represent INF- y, IL-17A, and TNF-a, respectively, and the results showed that the peroxide reductase IV antigen stimulates RA PBMC secretion in vitro and is associated with RA pathogenesis, IL-17A. , TNF-a and INF- γ. PBMCs that do not stimulate sputum, normal people, and other autoimmune diseases such as SLE secrete these cytokines.

Conclusion: Peroxiredoxin IV antigen specifically stimulates the secretion of IL-17A, TNF-a and INF- Y by PBMC in RA patients. IL-17A, TNF-a and INF-Y are important cytokines involved in the development of RA disease, indicating that reductase IV is a RA-specific disease-associated antigen.

The preparation of vaccines based on the modification of the peroxide reductase IV antigen on the basis of the above examples may provide a new means for the specific immunotherapy of RA. It is to be understood that the above embodiments are merely illustrative of the embodiments of the present invention, and are not intended to be The various changes and modifications of the invention are intended to be

Claims

Claim

1. Use of peroxide reductase IV in the preparation of rheumatoid arthritis-specific antigens.

The use according to claim 1, characterized by the use of the peroxide reductase IV antigen, epitope peptide, immune complex and antibody thereof for preparing a rheumatoid arthritis diagnostic reagent.

The use according to claim 2, characterized in that the use of the peroxidase IV antigen, the epitope peptide, the immune complex and the antibody thereof for preparing an early diagnostic reagent for rheumatoid arthritis.

4. Use according to claim 1, characterized by the use of peroxide reductase IV in the preparation of a rheumatoid arthritis vaccine.